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16-56
Mutagens Alter DNA Structure in
Different Ways

Chemical mutagens come into three main types

1. Base modifiers

2. Intercalating agents

3. Base analogues
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16-57

Base modifiers covalently modify the structure of a nucleotide
 For example, nitrous acid, replaces amino groups with
keto groups (–NH2 to =O)
 This can change cytosine to uracil and adenine to
hypoxanthine

These modified bases do not pair with the appropriate nucleotides
in the daughter strand during DNA replication
These mispairings
create mutations in the
newly replicated strand

Some chemical mutagens disrupt the appropriate pairing
between nucleotides by alkylating bases within the DNA

Examples: Nitrogen mustards and ethyl methanesulfonate (EMS)

Intercalating agents contain flat planar structures
that intercalate themselves into the double helix



This distorts the helical structure
When DNA containing these mutagens is replicated, the
daughter strands may contain single-nucleotide additions
and/or deletions resulting in frameshifts
Examples:


Acridine dyes
Proflavin
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16-60

Base analogues become incorporated into daughter
strands during DNA replication

For example, 5-bromouracil is a thymine analogue

It can be incorporated into DNA instead of thymine
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H
O
Br
N
H
H
N
O
5-bromouracil
(keto form)
O
N
N
Sugar
Sugar
H
H
H
N
O
O
This tautomeric shift
occurs at a relatively
high rate
H
H
N
N
N
5-bromouracil
(enol form)
Adenine
Normal pairing
Figure 16.16
Br
H
N
N
N
Sugar
N
N
Sugar
N
H
Guanine
Mispairing
(a) Base pairing of 5BU with adenine or guanine
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16-61
In this way, 5-bromouracil can promote a change
of an AT base pair into a GC base pair
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5′
5′
3′
A 5BU
3′
3′
A T
DNA
replication
3′
5′
5′
5′
5′
3′
G 5BU
3′
5′
3′
G C
DNA
replication
3′
5′
5′
3′
G or A 5BU
3′
5′
(b) How 5BU causes a mutation in a base pair during DNA replication
Figure 16.16
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16-62

Physical mutagens come into two main types



1. Ionizing radiation
2. Nonionizing radiation
Ionizing radiation





Includes X-rays and gamma rays
Has short wavelength and high energy
Can penetrate deeply into biological molecules
Creates chemically reactive molecules termed free radicals
Can cause





Base deletions
Oxidized bases
Single nicks in DNA strands
Cross-linking
Chromosomal breaks
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16-63
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H
O

Nonionizing radiation





Includes UV light
Has less energy
Cannot penetrate deeply
into biological molecules
Causes the formation of
cross-linked thymine
dimers
Thymine dimers may
cause mutations when that
DNA strand is replicated
O
P
O
CH2
O–
H
H
H
N
P
CH3
H
H
Thymine
CH3
O
O
O
CH2
O–
H
H
O
O
H
H
N
N
H
H
O
Thymine
H
Ultraviolet
light
O
O
P
O
O
H
O
CH2
O–
H
H
N
O
O
O
H
H
N
H
CH3
H
H
O
O
P
O
CH2
O–
Figure 16.17
N
O
H
H
CH3
H
O
O
H
N
H
H
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N
H
O
Thymine dimer
16-64
Testing Methods Can Determine If an
Agent Is a Mutagen

Many different kinds of tests have been used to
evaluate mutagenicity

One commonly used test is the Ames test


Developed by Bruce Ames
The test uses a strain of Salmonella typhimurium that cannot
synthesize the amino acid histidine



It has a point mutation in a gene involved in histidine biosynthesis
A second mutation (i.e., a reversion) may occur restoring the
ability to synthesize histidine
The Ames test monitors the rate at which this second mutation
occurs
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16-65
Mix together the Salmonella strain, rat liver extract, and suspected
mutagen. The suspected mutagen is omitted from the control sample.
Provides a
mixture of
enzymes that
may activate a
mutagen
Control
Suspected
mutagen
Rat liver
extract
Rat liver
extract
Salmonella
strain
(requires
histidine)
Salmonella
strain
(requires
histidine)
Plate the mixtures onto petri
plates that lack histidine.
Incubate overnight to
allow bacterial growth.
The control plate
indicates that
there is a low
level of
spontaneous
mutation
A large number of colonies
suggests that the suspected
mutagen causes mutation.
Figure 16.18 The Ames test for mutagenicity
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16-66
16.3 DNA REPAIR
• Since most mutations are deleterious, DNA repair
systems are vital to the survival of all organisms
– Living cells contain several DNA repair systems that can
fix different type of DNA alterations
• In most cases, DNA repair is a multi-step process
– 1. An irregularity in DNA structure is detected
– 2. The abnormal DNA is removed
– 3. Normal DNA is synthesized
• Refer to Table 16.7
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16-67
16-68
Damaged Bases Can Be Directly Repaired

In a few cases, the covalent modifications of
nucleotides can be reversed by specific enzymes

Photolyase can repair thymine
dimers


It splits the dimers restoring the
DNA to its original condition
Uses energy of visible light

O6-alkylguanine alkyltransferase repairs alkylated bases

It transfers the methyl or ethyl group from the base to a cysteine
side chain within the alkyltransferase protein

Surprisingly, this permanently inactivates alkyltransferase!
Base Excision Repair Removes a
Damaged DNA

Base excision repair (BER) involves a category of
enzymes known as DNA N-glycosylases


Depending on the species, this repair system can
eliminate abnormal bases such as



These enzymes can recognize an abnormal base and
cleave the bond between it and the sugar in the DNA
Uracil; Thymine dimers
3-methyladenine; 7-methylguanine
Refer to Figure 16.20
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16-71
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5′
3′
C
G
G
C
T
C
A
G
A
T
A
U
C
G
G
C
A
T
C
G
G
C
A
T
C
C
T
G
G
A
3′
5′
N-glycosylase recognizes an abnormal
base and cleaves the bond between the
base and the sugar.
U
5′
3′
C
G
G
C
T
C
A
G
A
T
A
C
G
G
C
A
T
C
G
G
C
A
T
C
C
T
G
G
A
3′
5′
Apyrimidinic
nucleotide
AP endonuclease recognizes a missing
base and cleaves the DNA backbone on
the 5′ side of the missing base.
5′
3′
G
C
C
T
C
G
A
G
A
A
T
C
G
G
A
C
T
C
G
G
A
C
T
C
C
T
G
G
A
3′
5′
Depending on whether a purine
or pyrimidine is removed, this
creates an apurinic and an
apyrimidinic site, respectively
Nick
In E. coli, DNA polymerase I uses its 5′
3′
exonuclease activity to remove the damaged
region and then fills in the region with normal
DNA. DNA ligase seals the region.
5′
3′
G
C
C
G
T
A
C
G
A
A
T
T
C
G
G
A
C
T
C
G
G
A
C
T
C
G
C
G
T
A
3′
In eukaryotes such as humans, DNA
polymerase β can remove the apyrimidinic
nucleotide and replace it with the correct
nucleotide. DNA ligase seals the region.
5′
Nick-translated region
5′
3′
G
C
C
T
C
G
A
G
A
A
T
T
C
G
G
A
C
T
C
G
G
A
C
T
C
C
T
G
G
A
In eukaryotes such as humans,
DNA polymerase δ or ε can
synthesize a short segment of
DNA, which generates a flap.
5′
3′
5′
3′
G
C
C
T
C
G
A G
A
A
T
T
C
G
G
A
C
T
C
G
G
A
C
T
C
C
T
G
G
A
3′
5′
Flap is removed by flap
endonuclease. DNA ligase
seals the region.
Flap
5′
3′
G
C
Figure 16.20
Base Excision Repair
3′
C
G
T
A
C
G
A
A
T
T
C
G
G
A
C
T
C
G
G
A
C
T
C
G
C
G
T
A
5′
16-72
Nucleotide Excision Repair Removes
Damaged DNA Segments


An important general process for DNA repair is
nucleotide excision repair (NER)
This type of system can repair many types of DNA
damage, including



Thymine dimers and chemically modified bases
missing bases, some types of crosslinks
NER is found in all eukaryotes and prokaryotes

However, its molecular mechanism is better understood in
prokaryotes
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16-73
Nucleotide Excision Repair Removes
Damaged DNA Segments

In E. coli, the NER system requires four key proteins

These are designated UvrA, UvrB, UvrC and UvrD

Named as such because they are involved in Ultraviolet light repair
of pyrimidine dimers


They are also important in repairing chemically damaged DNA
UvrA, B, C, and D recognize and remove a short segment
of damaged DNA

DNA polymerase and ligase finish the repair job

Refer to Figure 16.21
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16-74
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Thymine dimer
5′
3′
A
3′
T T
A
5′
B
The UvrA/UvrB complex tracks along the
DNA in search of damaged DNA.
5′
3′
A
T T
3′
A
B
5′
After damage is detected, UvrA
is released, and UvrC binds.
5′
3′
T T
3′
B
5′
UvrC
UvrC makes cuts on both
sides of the thymine dimer.
Figure 16.21
16-75
Typically, the cuts are 4-5 nucleotides from the 3’ end of the damage,
and 8 nucleotides from the 5’ end
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Cut
Cut
5′
3′
T T
3′
B
5′
UvrC
UvrD, which is a helicase, removes
the damaged region. UvrB and
UvrC are also released.
5′
3′
3′
5′
DNA polymerase fills in the gap,
and DNA ligase seals the gap.
No thymine dimer
Figure 16.21
5′
3′
3′
5′
16-76
Nucleotide Excision Repair Removes
Damaged DNA Segments

Several human diseases have been shown to
involve inherited defects in genes involved in NER

These include xeroderma pigmentosum (XP), Cockayne
syndrome (CS) and PIBIDS


A common characteristic of all three
syndromes is an increased sensitivity to
sunlight
Xeroderma pigmentosum can be
caused by defects in seven different
NER genes
Mismatch Repair Systems Detect and
Correct A Base Pair Mismatch


A base mismatch is another type of abnormality in
DNA
The structure of the DNA double helix obeys the
AT/GC rule of base pairing


However, during DNA replication an incorrect base may be
added to the growing strand by mistake
DNA polymerases have a 3’ to 5’ proofreading ability
that can detect base mismatches and fix them
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16-78
Mismatch Repair Systems Detect and
Correct A Base Pair Mismatch



If proofreading fails, the mismatch repair system
comes to the rescue
Mismatch repair systems are found in all species
An important aspect of these systems is that they are
specific to the newly made strand
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16-79
Mismatch Repair Systems Detect and
Correct A Base Pair Mismatch

The molecular mechanism of mismatch repair has
been studied extensively in E. coli

Three proteins, MutL, MutH and MutS detect the mismatch
and direct its removal from the newly made strand


The proteins are named Mut because their absence leads to a
much higher mutation rate than normal
A key characteristic of MutH is that it can distinguish
between the parental strand and the daughter strand


Prior to replication, both strands are methylated
Immediately after replication, the parental strand is methylated
whereas the daughter is not!
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16-80
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The MutS protein finds a mismatch. The MutS/MutL complex binds
to MutH, which is already bound to a hemimethylated sequence.
m
GATC
C TAG
Parental
strand
MutH
Newly
made
strand
MutL
Acts as a linker between
MutS and MutH
MutS
T
G
Incorrect
base
MutH makes a cut in the
nonmethylated strand. MutU
separates the DNA strands at the
cleavage site and an exonuclease
digests the nonmethylated strand
just beyond the base mismatch.
m
T C
GA
MutH cleavage site
G
Figure 16.23 Methyl-directed mismatch repair in E. coli
16-81
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m
T C
GA
MutH cleavage site
G
DNA polymerase fills in
the vacant region. DNA
ligase seals the ends.
m
GATC
C TAG
The mismatch has been
repaired correctly.
C
G
Figure 16.23 Methyl-directed mismatch repair in E. coli
16-82
Double-Strand Breaks in DNA Can Be
Repaired by Recombination

DNA Double-Strand Breaks are very dangerous


Breakage of chromosomes into pieces
Caused by ionizing radiation and chemical mutagens




Also caused by reactive oxygen species which are the byproducts of
cellular metabolism
10-100 breaks occur each day in a typical human cell
Breaks can cause chromosomal rearrangements and
deficiencies
They may be repaired by two systems known as
homologous recombination repair (HRR) and
nonhomologous end joining (NHEJ)

Refer to Figures 16.24 and 16.25
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16-83
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Double-strand break
5′
3′
3′
An identical
3′
region
between
sister
5′
chromatids
5′
5′
3′
End processing
5′
3′
3′
5′
3′
5′
5′
3′
Strand exchange
Figure 16.24
5′
3′
3′
5′
3′
5′
5′
3′
16-81
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5′
3′
3′
5′
3′
5′
5′
3′
DNA synthesis
5′
3′
3′
5′
3′
5′
5′
3′
Resolution and ligation
Figure 16.24
5′
3′
3′
5′
3′
5′
5′
3′
16-85
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Double-strand break
End binding
End-binding proteins
End bridging
Protein cross-bridge
Recruitment of additional
proteins and end processing
Proteins for DNA processing
Gap filling and ligation
Figure 16.25
16-86
Repair of Actively Transcribed DNA

Not all DNA is repaired at the same rate


Actively transcribed genes in eukaryotes and prokaryotes
are more efficiently repaired than is nontranscribed DNA
The targeting of DNA repair enzymes to actively
transcribing genes has several biological advantages

Active genes are more loosely packed



May be more vulnerable to DNA damage
Transcription may make DNA more susceptible to damage
DNA regions that contain active genes are more likely to
be important for survival than nontranscribed regions
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16-87

In E. coli, a protein
known as
transcription-repair
coupling factor
(TRCF) mediates
transcription coupled
DNA repair

It targets the NER
system to actively
transcribed genes with
damaged DNA


In eukaryotes, the mechanism that couples DNA
repair and transcription is not completely understood
Several different proteins have been shown to act as
transcription-repair coupling factors


Some of these have been identified in people with high
rates of mutation
For example, in Cockayne syndrome

Two genes, CS-A and CS-B, encode proteins that function as
transcription-repair coupling factors
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16-90
Damaged DNA May Be Replicated
by Translesion DNA Polymerases

It is inevitable that some lesions may escape all
repair systems


Such lesions may be present when DNA is replicated
Replicative DNA polymerases, such as DNA pol III in
E. coli, are sensitive to geometric distortions in DNA


They are unable to replicate through DNA lesions
Indeed, this type of replication requires specialized DNA
polymerases
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16-91


These specialized enzymes assist the replicative
DNA pol in the translesion synthesis (TLS) process
The TLS polymerases contain an active site with a
loose, flexible pocket


They can accommodate aberrant structures in the template
strand
A negative consequence of TLS polymerases is their low
fidelity


The mutation rate is typically in the range of 10-2 to 10-3 (errorprone replication)
When a replicative DNA pol encounters a damaged
region, it is swapped for a TLS polymerase
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16-92


In E. coli, translesion synthesis occurs under
extreme conditions that promote damage to DNA
This is termed the SOS response


It results in the up-regulation of several genes that repair
DNA, restore replication and prevent premature division
The damaged DNA that has not been repaired is
replicated by DNA polymerases II, IV and V
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16-93